Polarization based sensing

ABSTRACT

The present invention relates to the determination of the presence or concentration of an analyte in a sample by visual or electronic element, using polarization based sensing techniques ( 14 ) employing fluorescent sensing ( 11 ) and reference molecules ( 10 ).

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of application Ser. No. 60/107,997filed Nov. 11, 1998.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The work described herein was supported by a grant from the NationalInstitutes of Health National Center for Research Resources RR-08119.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the determination of the presence orconcentration of an analyte in a sample by visual or electronic means,using polarization based sensing techniques employing fluorescentsensing and reference molecules.

2. Description of the Related Art

A bibliography follows at the end of the Detailed Description of theInvention. The listed references are all incorporated herein byreference.

During the past ten years there have been remarkable advances in thetechnology for fluorescence sensing [1-7]. There has been extensivedevelopment of new fluorescent probes [8-10], and the introduction oftime-resolved fluorescence to chemical sensing, which is referred to aslifetime-based sensing [11-13]. Additionally, the timescale offluorescence has been extended from the nanosecond range to themicrosecond range by the use of long-lifetime metal-ligand complexes[14-15].

New approaches to fluorescence sensing continue to appear. Recently anew approach to sensing has been developed which uses referencefluorophores in addition to the sensing fluorophores. The concept is tomix a sensing fluorophore which is sensitive to an analyte with a secondfluorophore which is not sensitive to the analyte. One then measures thecombined emission of the sensor and reference fluorophores, which can beused to determine the analyte concentration. This approach has been usedwith the long lifetime metal-ligand complexes (MLC) as the referencefluorophore, and a pH sensitive probe, to determine pH or pCO₂ from thephase angle of the emission [16-17]. The present inventors have alsoused such mixtures to determine pH, calcium, and glucose concentrations.In our studies we used the low frequency modulation of the emission,rather than the phase angle, to determine the analyte concentration[18-21]. We showed that the low frequency modulation can be used todetermine the fractional intensity of the nanosecond fluorophore,relative to that of the metal-ligand complex with its microsecond decaytime.

In a recently published paper [22], the present inventors extended theidea of using a reference fluorophore to sensing based on anisotropymeasurements. The concept is based on the additivity of anisotropy[23-25]. This rule states that the anisotropy for a mixture offluorophores is the weighted average of the value for each fluorophoreand their fractional contributions to the total intensity. Thus, anintensity change of the sensing fluorophore is transformed into a changein anisotropy or polarization by appropriate placement of polarizers. Wedeveloped sensing methods in which the reference was a fluorophore in astretch-oriented film of polyvinyl alcohol. We used anisotropy-basedsensing to measure pH using 6-carboxy fluorescein or the concentrationof labeled protein in the sample.

However, there remains a need in the art for improved methods fordetermining the presence or concentration of an analyte usingfluorescent reference and sensing molecules.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a method for determiningthe presence or concentration of an analyte, comprising the steps of:

a) providing a fluorescent reference molecule and a fluorescent sensingmolecule;

b) exposing said sensing molecule to an analyte to form a mixture,wherein said analyte is capable of changing the intensity of thefluorescence emitted by the sensing molecule in aconcentration-dependent manner;

c) exposing said reference molecule and said. mixture to a radiationsource which causes said reference and sensing molecules to emitfluorescence;

d) polarizing said emitted fluorescence through two differentpolarization axes which are substantially perpendicular to each other;

e) attenuating the emission from one of the polarization axes, ifnecessary, such that the intensities of the emissions through both axesare substantially equal; and

f) correlating the degree of attenuation with the presence orconcentration of said analyte in said sample.

In another aspect, the present invention relates to a sensor fordetermining the presence or concentration of an analyte in a sample,which comprises:

a) a fluorescent reference molecule;

b) a fluorescent sensing molecule, wherein said analyte is capable ofchanging the intensity of the fluorescence emitted by the sensingmolecule in a concentration-dependent manner;

c) optionally a radiation source which is capable of causing saidreference and sensing molecules to emit fluorescence;

d) means for isolating said emitted fluorescence along two differentpolarization axes which are substantially perpendicular to each other;and

e) means for attenuating the emission from one of the polarization axes,such that the intensities of the emissions through both axes aresubstantially equal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the calculated compensation angles for in-line anisotropysensing (FIG. 1A) and front-face anisotropy sensing (FIGS. 1B and 1C).The lower two panels are similar, except for the scale of the x-axis.

FIG. 2 shows the dependence of the intensity ratio for change in α of 1,2 or 3 degrees. The x-axis is the starting angle α₀. The intensity ratiowas plotted as a value greater than unity.

FIG. 3 shows the absorption and emission spectra of the referencefluorophore MPSPI in an unoriented polyvinyl alcohol film. Also shownare the excitation and emission anisotropy spectra in the PVA film. Theexcitation wavelengths available from the LED and HeNe laser areindicated on the x-axis.

FIG. 4 shows the fluorescence polarization of MPSPI in the PVA film as afunction of the stretching ratio. The stretching ratio R_(S) is relatedto the actual physical fold of the stretch N by R_(S)=N^(3/2) [29].

FIG. 5 shows the emission spectra of rhodamine B in ethanol with (-) andwithout ( . . . ) with the MPSPI reference. The dashed line (- - -)shows the emission spectrum of MPSPI alone. The additional dotted lineshows the transmission profile of the emission filter.

FIG. 6 shows the emitted light observed through the analyzer polarizer(P) for different concentrations of rhodamine B using the MPSPIreference and the in-line geometry. The position of the analyzerpolarizer is at α₀ near 75° for all images. The listed values are thecompensation angles (Δα) needed to equalize the intensities.

FIG. 7 shows the dependence of the compensation angle (Δα) on theconcentration of rhodamine B using the in-line geometry. The uncertaintyin the compensation angle is shown as ΔΔα.

FIG. 8 shows the dependence of the compensation angle on the rhodamine Bconcentration in 0.5% intralipid. These data were obtained using thefront-face geometry. The insert shows the emission spectra observed forthe rhodamine B-MPSPI sample. The dashed line emission spectrum is ofthe MPSPI reference alone.

FIG. 9 shows the emission spectra of a front-face anisotropy pH sensorbased on 6-carboxyfluorescein. The dashed line shows the transmissionprofile of the emission filter used for the visual measurements.

FIG. 10 shows the compensation angles for the front-face polarization pHsensor.

FIG. 11 shows the visual detection of the concentration of [Ru(bpy)₃]²⁺measured using the same compound [Ru(bpy)₃]²⁺ as the reference. ELL,electroluminescent light source; F_(ex), excitation filter; R, referencesolution with a constant concentration of [Ru(bpy)₃]²⁺, P_(⊥),polarizer; S, sample with varying concentrations of [Ru(bpy)₃]²⁺,F_(em), emission filter; DP, dual polarizer; P, analyzer polarizer.

FIG. 12A depicts one embodiment of a blood chemistry device according tothe present invention. The excitation source, sample and detector are inan in-line geometry. FIG. 12B depicts another device which analyses datafrom an implantable patch. The fluorescence from the implanted patchand/or tissue is observed using front-face geometry.

FIG. 13 depicts two optical systems for anisotropy-based sensing withvisual detection. FIG. 13A depicts an in-line geometry with a stretchedfilm. FIG. 13B depicts a front-face geometry. In the front-face geometrywith the stretched film it is possible to use an additional polarizerP_(⊥), which allows selective detection of horizontal component of thefluorescence from the sample cuvette K. Such a configuration extends therange of angles (angles needed to equalize the transmittance of bothpolarizers in DP) to 90°. Front-face anisotropy sensing can be performedwithout the additional polarizer, resulting in the 45° range of angles.

FIG. 14 depicts polarizer angles for in-line anisotropy sensing. In FIG.14A there is no emission from the sample. For a strongly orientedreference the initial polarizer angle is near 90° from the vertical. InFIG. 14B the sample emission is dominant, and the angle is near 45° fromthe vertical.

FIG. 15 depicts various polarizer angles for front-face anisotropysensing. In FIG. 15A there is no emission from the sample. For astrongly oriented reference film the initial polarizer angle is near 90°from the vertical. In FIG. 15C the sample emission is dominant, and theangle approaches 0° from the vertical.

FIG. 16 schematically depicts an apparatus for polarization sensing withvisual detection according to the present invention. The value of α is0° when the analyzer polarizer is oriented vertically.

FIGS. 17A and B are images seen through the analyzer for differentconcentrations of glucose (FIG. 17A) and calcium (FIG. 17B). In eachcase the initial angle of the analyzer was adjusted to yield equalintensities in the absence of glucose on calcium. The images were thenrecorded at the same analyzer angle. The values under the images are theanalyzer angles needed to equalize the intensities, not the angle neededto equalize the intensities.

FIG. 18 depicts the calibration curve for glucose sensing with visualdetection.

FIGS. 19A and B depict the polarization sensing of calcium using Fluo-3.FIG. 19A shows the emission spectra of the vertical component and of thehorizontal component. The calcium concentration is constant at 1.35micro molar in the left side of the sensor. The calcium concentration isvariable in the right side of the sensor. FIG. 19B shows thepolarization across the emission spectra.

FIG. 20 depicts the calibration curve for calcium sensing with visualdetection.

FIG. 21 schematically depicts an apparatus for polarization sensing withphotocell detection according to present invention. The value of a is 0°with the analyzer polarizer is oriented vertically.

FIG. 22 schematically depicts an apparatus for polarization sensing withphotocell detection according to the present invention. The expandedview on the right side of the figure shows optical components. Theexpanded view on the left side of the figure shows the detector withbridged photocells. The polarizer P is rotated until the signal on thevolt meter is null.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a new approach to fluorescence sensingwhich optionally relies on visual determination of the polarization andfluorescent reference and sensing molecules.

The present invention may be used to detect any analyte for which asuitable fluorescent sensing probe is available, i.e., a sensingmolecule whose fluorescent emission changes upon exposure to the analytein a concentration-dependent manner. Many such molecules are well-known,and may be used to measure pH, glucose, oxygen, blood gases, variousions, proteins, etc.

The fluorescent reference molecule is chosen to provide a constantreference or background. There are a wide range of commerciallyavailable suitable reference molecules, for example those sold byMolecular Probes, Inc., Eugene, Oreg. and other companies. The referencemolecule may be a distinct entity but having the same structure as thesensing molecule, provided that the reference molecule is not exposed tothe analyte, e.g., is isolated in a separate compartment or the like. Ina preferred embodiment, the reference molecule is embedded in anoriented film which is not effected by the analyte.

In practice, the sensing molecule is exposed to a sample containing ananalyte of interest. The reference and sensing molecules are thenexposed to a radiation source which causes the molecules to emitfluorescence. The choice of the radiation source will depend on a numberof factors, such as the fluorescent characteristics of the reference andsensing molecules, the specific application, etc. Preferred sourcesinclude HeNe lasers, blue LEDs, sunlight and room light.

The fluorescent emission from the reference and sensing molecules isthen polarized along two substantially perpendicular polarization axes.That is preferably accomplished by allowing the emission from eachmolecule to pass through a dual polarizer, with adjacent sectionsoriented substantially orthogonally to each other. Optionally, beforepassing through the dual polarizer, the emitted fluorescence is allowedto pass through a filter which substantially eliminates scatteredexcitation.

After the emission is isolated along the two polarization axes, it willgenerally be the case that the two emissions will have differentintensities. In the next step, the intensities are substantiallyequalized. That may be accomplished by, e.g., allowing the output fromthe dual polarizer to pass through an analyzer polarizer, which may berotated until both intensities are substantially equal. Thedetermination of when the intensities are substantially equal in thesimplest case may be made visually, or it may be made electronically bymeans which would be readily apparent to those skilled in the art. Forexample, the intensities may be measured in independent detectors, andthe analyzer polarizer may be adjusted until the signals are equal.Alternatively, one could use the ratio of two signals and adjust theratio to unity. In still another embodiment, the output from the twodetectors could be used in a balanced circuit, such as a Wheatstonebridge. The degree of rotation needed, relative to an initial referenceposition (for the emission from the reference molecule in the absence ofthe sensing molecule) is then correlated with the presence orconcentration of the analyte. One suitable electronic detector isdepicted in FIGS. 21 and 22, in which the apparatus comprises filter 12,containers 10 and 11 which house the reference and sensing molecules,filter 13, analyzer polarizer 14, and dual photocell 15. In FIGS. 21 and22, container 10 has on its surface a polarizing film orientedvertically, and container 11 has on its surface a polarizing filmoriented horizontally.

The foregoing approach has used to measure the concentration of RhB inintralipid, and to measure pH using 6-carboxy-fluorescein. The analyzerangle is typically accurate to one degree, providing pH values accurateto ±0.1 pH unit at the mid-point of the titration curve.

The present invention further relates to a method of visual polarizationsensing which does not require an oriented film, and which can use thesame fluorophore for the sample and reference. These approaches tovisual sensing are generic and can be applied to a wide variety ofanalytes for which fluorescent probes are available. Importantly, thedevices are simple, with the only electronic component being the lightsource.

Theory—In-Line Geometry

In the biophysical use of fluorescence it is common to use theanisotropy of the emission. However, in connection with the presentinvention it is possible to visually observe the emission from the frontsurface (front-face geometry) or directly through (in-line) the sample(in-line geometry). For these geometric conditions it is easier todescribe the results in terms of the polarization of the emission,$\begin{matrix}{P = \frac{I^{\parallel} - I^{\bot}}{I^{\parallel} + I^{\bot}}} & (2)\end{matrix}$

where I^(∥) and I^(⊥) mare the intensities seen parallel (∥) orperpendicular (⊥) to the orientation of the reference film.

The theory for visual detection of the polarization can be developedusing FIG. 14. This scheme shows the various polarized intensities fromthe sample (S) and reference (R) which are present along the opticalpath. Suppose the sample is illuminated with unpolarized light. Theemission from the stretched film is polarized along its stretch axis(I_(R) ^(∥)). Since the alignment is never perfect, there is also asmaller component perpendicular to the stretch axis (I_(R) ^(∥)). Thestarting angle of the polarizer measured from the vertical axis (α₀)will be near 90°. The angle will be near 90° because the film emissionis highly polarized, and the vertical component (I_(R) ^(⊥)) must besignificantly attenuated to yield equal intensities in each half of thedual polarizer.

Suppose now there is significant emission from the sample (FIG. 14,lower panel). Since the excitation is not polarized, and the sample isnot oriented, the sample adds components of equal intensity in bothdirections (I_(S) ^(∥)=I_(S) ^(⊥)) We have assumed that the stretchedfilm is optically thin, so that it does not act like a polarizer. Theemission transmitted through each side of the dual polarizer is the sumof the two polarized components

I^(∥)=I_(R) ^(∥)+I_(S) ^(∥)  (3)

I^(⊥)=I_(R) ^(⊥)+I_(S) ^(⊥).  (4)

Assume now that these two components are observed through the analyzerpolarizer. The intensities observed through the vertical (V) andhorizontal (H) regions of the dual polarizer (DP) are given by

I^(V)=(I_(R) ^(∥)+I_(S) ^(∥))cos² α  (5)

I^(H)=(I_(R⊥)+I_(S) ^(⊥))sin² α  (6)

where α is the analyzer polarizer angle from the vertical position.

For visual measurement the analyzer is rotated until the intensity isequal for both sides of the dual polarizer. For this condition one has

(I_(R) ^(∥)+I_(S) ^(∥))cos² α(I_(R) ^(⊥)+I_(S) ^(⊥))sin^(2 α)  (7)

and $\begin{matrix}{{\tan^{2}\alpha} = {\frac{I_{R}^{\parallel} + I_{S}^{\parallel}}{I_{R}^{\bot} + I_{S}^{\bot}}.}} & (8)\end{matrix}$

The stretched film displays a constant polarization value, which can bedefined in terms of the ratio of the polarized intensities,

k=I_(R) ^(∥)/I_(R) ^(⊥.)  (9)

For an isotropic film there is no polarization and k=1. Hence theinitial angle for the analyzer polarizer is given by tan² α₀=1.0 orα₀=45°. For a perfectly oriented film k=infinity, tan² α₀ is very largeso that α₀ is near 90°. In practice one obtains highly but imperfectlyoriented samples with values of k ranging from 10 to 12 in the case ofisotropic excitation.

It is instructive to examine how the polarizer angle depends on therelative fluorescence intensity from the sample and the reference. Letthe total intensity from the reference be defined as

I_(R) ^(T)=I_(R) ^(∥)+I_(R) ^(⊥).  (10)

Dividing the numerator and denominator in eq. 8 by I_(R) ^(T) yields$\begin{matrix}{{\tan^{2}\alpha} = {\frac{\frac{k}{k + 1} + \frac{I_{S}^{\parallel}}{I_{R}^{T}}}{\frac{1}{k + 1} + \frac{I_{S}^{\bot}}{I_{R}^{T}}}.}} & (11)\end{matrix}$

For many situations the emission from the sample will be unpolarized,I_(S) ^(∥)=I_(S) ^(⊥). For this condition one can define the ratio n,which is the ratio of the total emission from the sample to that of thereference, $\begin{matrix}{n = {\frac{I_{S}^{T}}{I_{R}^{T}} = {\frac{I_{S}^{\parallel} + I_{S}^{\bot}}{I_{R}^{T}}.}}} & (12)\end{matrix}$

Introduction of this ratio into eq. 11 yields $\begin{matrix}{{\tan^{2}\alpha} = {\frac{\frac{k}{k + 1} + \frac{n}{2}}{\frac{1}{k + 1} + \frac{n}{2}}.}} & (13)\end{matrix}$

This expression (eq. 13) describes the angle of the polarizer needed toequalize the intensities in terms of the polarization ratio of thereference (k) and the relative intensity of the sample to that of thereference (n).

It is informative to examine the range of polarizer angles which canoccur as the intensity of the sample increases. The initial condition(α₀) is found when there is no emission from the sample, so that all thesignal originates with the reference film. If the emission from the filmwere completely polarized (k=∞) then the analyzer would need to beoriented at 90° to equalize the intensities to zero. Basically, onewould have to nearly extinguish the signal I_(R) ^(∥) by orienting theanalyzer nearly perpendicular to the film axis.

In practice, the emission from the film is not completely polarized, butis defined by a finite value of the k value. For a typical value of k=12the initial angle α₀ with no fluorescence from the sample is about 74°,which can be found using eq. 13 with n=0. Alternatively, one cancalculate α₀ using

tan α₀={square root over (k)}.  (14)

Now consider the condition when the emission from the sample is thedominant emission. We also assume that the sample emission is notpolarized. Under these conditions the intensities I^(V) and I^(H) willbe equal yielding tan² α=1.0. The intensities seen through the analyzerwill be equal when the analyzer is rotated 45° from the vertical. Thisvalue can be found by noting that as n becomes much larger than k, tan aapproaches unity (eq. 13) so a approaches 45°.

It is convenient to examine the changes in polarizer angle (Δα) in theabsence and presence of sample. We call this value the “compensationangle.” This change in angle is given by $\begin{matrix}{{{\Delta\alpha} = {{\alpha_{0} - \alpha} = {{\arctan \sqrt{k}} - {\arctan {\sqrt{\frac{\frac{k}{k + 1} + \frac{n}{2}}{\frac{1}{k + 1} + \frac{n}{2}}}.}}}}}~} & (15)\end{matrix}$

Simulated values of ΔΔ are shown in FIG. 1A. These values werecalculated for various values of k, and for a 10-fold range of relativeintensities n=I_(S) ^(T)/I_(R) ^(T). In the absence of fluorescence fromthe sample the polarizer remains at the initial value near 90° withΔα=0. As the sample intensity increases the polarizer must be rotatedtowards 45° to equalize the intensities. For experimentally accessiblevalues of k near 12, the range of Δα values is about 30 degrees. Whenusing the in-line geometry without the additional polarizer (P_(⊥) inFIG. 13) the range of angles depends on the orientation (k) of the film.The film must be oriented to some extent (k>1) otherwise there is nochange in the compensation angle. More highly oriented films, or largervalues of k, yield larger changes in α.

Theory-Front Face Geometry

The operational principles of the front face polarization sensor (FIG.13, lower panel) can be understood by similar reasoning. The front facesensor can be used without the extra polarizer P_(⊥), in which case therange of angles is the same described above for the in-line geometry. Analternative approach is to use a polarizer P_(⊥) in front of the sample.This polarizer selects for the perpendicular component of the sampleemission. Under this condition, I_(S) ^(∥) is zero. Assuming the sampleemission is unpolarized, the total emission from the sample is I_(S)^(T)=I_(S) ^(⊥), and eq. 11 becomes $\begin{matrix}{{\tan^{2}\alpha} = {\frac{\frac{k}{k + 1}}{\frac{1}{k + 1} + n}.}} & (16)\end{matrix}$

The limiting conditions for the front face sensor can be understood fromFIG. 15. In the absence of emission from the sample the polarizer angleα₀ is defined by the value of k. For a perfectly aligned reference film(k=∞) the analyzer polarizer must be oriented near 90° to equalize theintensities (top). At intermediate sample intensities (middle panel) thevalue of a to equalize the intensities will be between 0 and 90°. Ifemission from the sample is the dominant emission, then most of theemitted light is horizontally polarized, and the polarization angle mustbe near zero to equalize the signals (lower panel). This can be seenfrom eq. 16. As n becomes large, tan a approaches zero, so a approacheszero. This result illustrates an important effect of using theadditional polarizer P_(⊥). The range of polarization angles doubles to90° as compared to the in-line geometry without the polarizer P_(⊥).

For the front-face sensor the compensation angles Δα are given by$\begin{matrix}{{{\Delta\alpha} = {{\alpha_{0} - \alpha} = {{\arctan \sqrt{k}} - {\arctan \sqrt{\frac{\frac{k}{k + 1}}{\frac{1}{k + 1} + n}}}}}}~} & (17)\end{matrix}$

Simulated values of Δα are shown in FIG. 1, middle and lower panels. Asthe sample intensity increases the value of Δα approaches 90°. Foravailable values of k near 12, Δα can be as large as 60°. Compared tothe in-line geometry it seems that a greater range of sample intensitiescan be measured using the front-face geometry than using the in-linegeometry (lower panel).

The simulated values of Δα for the front-face geometry reveal anotherpossibility, which is to perform anisotropy sensing without an orientedsample. This can be seen in FIG. 1 (middle and lower panels) for k=1,which is an unpolarized reference. A range of 45° is available usingeven an unpolarized reference. This possibility results from theadditional polarizer P_(⊥) which transmits only one polarized componentfrom the sample. Since the reference can be unpolarized, the referencecan consist of the sensing fluorophore itself, rather than a differentfluorophore in an oriented film.

Precision of Polarization Sensing with Visual Detection

The principle of polarization sensing with visual detection is toequalize the light intensities transmitted through both sides of thedual polarizer. In our initial experiments we were surprised to findthat the polarizer angle could be determined to within 1°. The relativeintensities change slowly for values of α near 45°, so that theprecision we observed seemed to be unexpectedly high. For this reason weperformed analyses to determine the expected precision of the αvalues.

The error in determining a should depend on the initial value of α(α₀),the accuracy of the rotary stage, and the sensitivity of our eyes todetect the intensity differences. Our rotary stage was accurate to 1°,and more accurate stages are readily available. Hence we considered theeffects of various values of α₀ and the eye sensitivity. From eq. 5 onecan show that the relative intensity through each side of the dualpolarizer is given by

I^(H)/I_(V)=tan² α.  (18)

We used this expression to calculate the relative change in intensityfor changes in α of 1, 2 or 3 degrees (FIG. 2). If α₀ is close to 0 or90° the intensity ratio is strongly dependent on the polarizer angle α.For example, a change in α from 45 to 46° changes the intensity ratio to1.072, and a change of two degrees, 45 to 47°, results in a relativeintensity change of 15%. Hence it appears that we must consider thesensitivity of the human eye to detecting relative intensities inadjacent images.

To answer the question of detectability of intensity differences weperformed the following tests. We used a high concentration of rhodamineB (RhB) in ethanol to obtain a starting value near 45°. Next we insertedquartz plates in front of one side of the dual polarizer. The measuredtransmission of these plates were 93.4, 87.3, 81.8 and 76.1%, for one,two, three and four plates, respectively.

How many plates were needed to obtain a detectable intensity differencebetween the two sides of the dual polarizer? We were surprised that mostof us could detect the presence of only one plate on either side of thedual polarizer. Everyone in the inventors' laboratory could recognizethe presence of two plates, which provides an intensity difference of1.14-fold or 14%. From these observations we judged that the averageindividual can detect an intensity ratio of 1.10, or a difference of10%. This suggests that the polarizer angle can be adjusted to about 1.4degrees in the worse case situation for α₀ near 45°.

We repeated these tests using a hand lamp and a red filter transmittingabove 590 nm, with no sample or reference solution. Once again we couldrecognize a difference due to just a single plate. For angles from 0 to25°, or from 65 to 90°, it is probable that the accuracy of thepolarizer angle can be greater than 1°. We did not perform such testsdue to the limited resolution of our rotary stage. In summary, theseresults indicate that the accuracy in the polarizer angle should be 1°under most experimental conditions. This accuracy will be shown below tobe adequate for typical sensing applications.

N-methyl-4-(pyrrolidinyl) styrylpyridinium iodide (MPSPI) was obtainedfrom Molecular Probes, Eugene, Oreg., rhodamine B (RhB) was fromExciton, Inc., Dayton, Ohio, and 6-carboxy fluorescein (6-CF) was fromEastman Kodak, Rochester, N.Y. Intralipid (20%) was obtained from KabiVitrum, Inc., Clayton, N.C. and diluted 40-fold to 0.5%, in 50 mM trisbuffer at the desired pH values.

Several excitation sources were used. We used a HeNe laser (543 nm) fromMeles Griot, or a blue LED from Nichia Chemical Industries, Tokushima,Japan. When using an LED an excitation bandpass of 466±26 nm [26] wasselected using a 510 nm short wavepass filter. We also used anelectroluminescent device, which was obtained from LumitekInternational, Inc., Ijamsville, Md. Its output was visually blue, witha maximum near 480 nm, and a half-width of 80 nm. Polarizing plasticfilms were from Rolyn Optics, Covina, Calif.

The fluorescence from RhB was observed through a 590 nm long pass glassfilter, and 6-CF fluorescein was observed through a 540 nm wide bandinterference filter from Chroma Technology Corp.

Films of polyvinyl alcohol were prepared as described previously[27-28]. These films were physically stretched up to 6-fold to orientthe MPSPI molecules and the film was then pressed against the side ofthe cuvette (FIG. 13). When using stretched films the stretching ratio(R_(S)) is defined as the axial ratio a/b of an ellipse which is formedwhen stretching an imaginary circle in the unoriented film [29]. Thevolume of the circle or ellipse is assumed to be conserved. Under theseconditions

R_(S)=N^(3/2)  (19)

where N is the physical fold of the stretch.

Stretched films provide an easily available reference fluorophore with ahigh polarization near unity. Such values can be obtained forfluorophores in stretched polymer films, which result in elongatedfluorophores being aligned along the stretching axis [27]. In suchsystems the electronic transitions of the fluorophore are all aligned inone direction, or more precisely display a uniaxial orientation. Theemission polarization from such samples are typically in the range of0.6 to 0.8, and can approach 1.0. [28-29]. Stretched polymer filmsretain their orientation for extended periods of time, and thus can bepractical for real-world applications.

Schematic diagrams of two possible geometries for visual polarizationsensing are shown in FIG. 13. In the in-line geometry the light passesthrough the sample. In the front-face geometry the emission from thesample is viewed from the illuminated surface. In both cases theabsorption of the stretched film is adjusted so that the exciting lightis only partially absorbed, and adequate intensity remains to excite thesample. A filter is used to eliminate the excitation and to transmit theemission.

An important part of the visual polarization sensor is the dualpolarizer (DP). This component consists of two adjacent sheet polarizerswith the optical axis of one rotated 90° relative to the other polarizer(FIG. 13). If the sample is uniformly illuminated, the intensitytransmitted by each half of the dual polarizer represents the parallel(∥) and perpendicular (⊥) components of the emission. This emission hastwo components, from the reference film and from the sample.

Visual detection of the degree of polarization may be accomplished byviewing the dual polarizer through an analyzer polarizer (P). Thispolarizer is rotated until the intensities are equal for both sides ofthe observation window. The total range of polarizer angles for thein-line geometry is 45°, as the emission ranges from 100% from thereference film to 100% from the sample. We found that it is easy tovisually detect the position of equal intensities. As will be shownbelow, visual observation was found to provide 1° of accuracy inadjusting the analyzer polarizer.

The operating principle is the same whether the geometry is in-line orfront face. However, the front-face geometry allows the use of anadditional polarizer (P_(⊥)) after the stretched film but before thesample. This polarizer (P_(⊥)) can be used to selectively observe onlythe perpendicular component of the emission from the sample. As will beshown below, the use of P_(⊥) increases the range of angles to 90°.

There are many advantages of polarization sensing with visual detection.One immediately obvious advantage is that a simple device may be used inpractice of the invention. The only electronic component is the lightsource. Excitation could be accomplished with LEDs, laser diodes orelectroluminescent devices, which may be powered using small batteries.Hence, such sensors will be extremely useful in emergency health care,doctor's offices and other medical applications. Additionally, thedevices may be sufficiently inexpensive to allow their use in lesscritical situations such as bioprocessing and process control.

It should be noted that the visual polarization sensor relies onintensity ratios, and will thus be sensitive to any factor that altersthe relative intensities of each polarized component. Hence thecalibration curve will depend on the concentration and/or intensity ofthe sensing and reference fluorophores. If one fluorophore photobleachesat a rate different from the other fluorophore, then the calibrationcurve will change. However, visual polarization sensors may be used withlarge area/low intensity illumination, which should minimizephotobleaching. Additionally, the inherent proximity focusing of thevisual polarization sensor will avoid the intensity changes which occurwith multiple optical components. Hence, a visual polarization sensorwill provide stable readings for extended periods of time.

Polarization of an Oriented Film

Prior to showing examples of polarization sensing it is valuable todiscuss the spectral properties of the reference film. We chose the dyeMPSPI because of its favorable absorption and emission spectra and itslarge Stokes' shift (FIG. 3). MPSPI can be excited with either the 543nm HeNe laser, or with the blue LED. Importantly, MPSPI displays a highfundamental anisotropy (r₀) across its absorption and emission spectra.

The elongated shape of MPSPI allows it to be strongly oriented instretched PVA films. This is shown in FIG. 4 which shows thefluorescence polarization as a function of the stretching ratio. As thestretching ratio increases the polarization increases to over 0.8. It isvaluable to notice that it is not necessary to use polarized excitation.The use of unpolarized excitation was mimicked by adjusting theexcitation polarization at 45° from the vertical. Prior to stretchingthe polarization is near zero for unpolarized (45°) excitation. However,for the stretched samples this polarization increases to over 0.8. Theincrease in polarization occurs irrespective of whether the film isexcited with vertically polarized or unpolarized light. Hence, polarizedemission from the reference film can be obtained without an excitationpolarizer.

The present invention will be further illustrated by means of thefollowing non-limiting examples.

EXAMPLE 1

Polarization Sensing of RhB

To characterize the polarization sensor we examined solutions ofrhodamine B (RhB) in ethanol at various RhB concentrations. Initially weuse the in-line geometry (FIG. 13, top). Emission spectra are shown inFIG. 5 for excitation at 543 nm. RhB displays a narrow emission with anemission maximum near 575 nm. The emission maximum of the MPSPIreference is at a similar wavelength, but the emission spectra of MPSPIis somewhat broader than RhB. As the RhB concentration increases, itsemission becomes dominant over that of the MPSPI reference. For thepolarization measurements the combined emission of MPSPI and RhB wasobserved using a filter which transmits above 580 nm.

FIG. 6 shows the visual images seen through the analyzer polarizer forincreasing concentrations of RhB. The polarization angle was initiallyadjusted to yield equal intensities in the absence of RhB. Since thereference film is highly polarized the initial value α₀ is near 75°. Allimages were recorded with the analyzer polarizer in this same position.As the RhB concentration increases, the intensities through each side ofthe dual polarizer become unequal. The direction of the intensitychanges can be understood by noting that the right side contains thehorizontal polarizer and the emission from RhB is unpolarized. Additionof RhB results in an increased relative intensity on the right side,where the dual polarizer and the analyzer polarizer are in thehorizontal position. The intensity increase is weaker on the left sidewhere the polarizers (DP and analyzer) are nearly crossed.

Next we examined the changes in the polarizer angle, the compensationangle, needed to equalize the intensities seen from each half of thedual polarizer (FIG. 7). To determine the compensation angle we measuredthe difference between the analyzer angles needed to equalize theintensities in the absence and presence of the RhB sample. As the RhBconcentration increased to 15 μM, the compensation angle increased byover 20° degrees. During these measurements we asked several members ofthis laboratory to adjust the polarizer and measure the difference angleΔα. Most individuals measured the same value of Δα to within 1 degree.It is interesting to consider the uncertainty in the RhB concentrationresulting from the 1° uncertainty in the compensation angle. At low RhBconcentrations the uncertainty in 0.5 μM, or about one part in four. Athigher RhB concentrations the uncertainty is about 1 μM, or one part inten. While this accuracy is somewhat poor, it is probably adequate forsome clinical determinations, particularly those which report a yes/noanswer rather than a specific value. We note that the range ofconcentrations is determined by the brightness of the reference film.Lower fluorophore concentrations could be measured if the reference filmis less fluorescent.

EXAMPLE 2

Fluorophores in Scattering Media

In many situations it is desirable to measure the fluorescence fromtissues, which can be due to intrinsic tissue fluorescence or due toextrinsic probes. Such measurements are becoming more important with thedevelopment of red or near infra-red (NIR) probes [30-32] and with theuse of these probes for trans-dermal measurements [33-34]. Hence wedecided to test the visual polarization sensor to measure theconcentration of RhB in 0.5% intralipid. Such solutions are highlyscattering, and mimic the scattering properties of skin.

As for the previous case, RhB and MPSPI were excited at 543 nm using aHeNe laser. In this case we used the front face geometry, as shown inFIG. 13 (lower panel), including the polarizer (P_(⊥)) in front of thesample. The compensation angles are shown in FIG. 8. Compared to theprevious data for the in-line geometry (FIG. 7), the front face geometryresults in a wider range of compensation angles, up to 30° or larger.This wider range is due to the additional polarizer (P_(⊥)) whichselected only the horizontal component of the RhB emission. We did notnotice any decrease in resolution due to the intralipid.

We note that it is not obvious that one can use polarizationmeasurements to measure fluorophore concentrations in scattering media.It is well known that light scattering results in depolarization of theemission from fluorophores within the scattering media. In the presentsituation this effect is not important because the polarization isimposed on the emission after it exits the scattering media.

EXAMPLE 3

Polarization Sensing of pH

As a practical illustration of polarization sensing we used 6-carboxyfluorescein (6-CF) as a pH-sensitive fluorophore. Fluorescein has beenwidely used as a pH-sensitive probe [35-37]. The pH-dependentfluorescence intensity is due to the carboxy group. Fluorescein ishighly fluorescent at higher pH values where this group is ionized, andmore weakly fluorescent at low pH. The intensity of fluorescein and itscarboxy derivatives increases dramatically over the pH range from 5 to8.

FIG. 9 shows the emission spectrum of fluorescein in the front-facegeometry, including the reference MPSPI film. In this case excitationwas accomplished with the 470 nm output of a blue light emitting diode(LED). This solid state light source can be powered by a 9 volt battery.The fluorescein emission at 525 nm increases about 5-fold from pH 5 to9. The emission of the MPSPI reference is at somewhat longerwavelengths. This illustrates one minor technical challenge in thedesign of a visual polarization sensor, the colors on each side of thedual polarizer can be slightly different due to the different relativeintensities of the fluorophore and the reference. In the present case weminimized these visual differences by selecting a relatively narrowrange of wavelengths for visual observation, from 540 to 560 nm. Inpractice it should not be difficult to obtain similar visual colors forthis sample and reference emission. A wide variety of fluorescentsensors are available [8], and a large number of fluorophores can beoriented in stretched films [27].

Compensation angles for the polarization pH sensor are shown in FIG. 10.These angles display the usual sigmoidal behavior for a pH sensor. Forthis initial visual pH sensor the one degree accuracy of thecompensation angle results in a pH accuracy to ±0.1 pH unit at thecenter of the titration curve. For clinical pH measurements the requiredaccuracy is ±0.02 [38-40]. Hence, the present sensor is not adequate foruse in blood gas measurements. However, an accuracy of ±0.1 pH unit isadequate in a wide range of less critical applications. We note that thepH accuracy is less than ±0.1 pH unit at pH values away from the centralpH value near 6.5. This is a characteristic of any optical indicatorwhich is based on a single dissociation constant.

It should be noted that the approach used for thefluorescein-polarization sensor can be applied to a wide variety ofanalytes. The only requirement is a fluorophore which changes intensityin response to the analyte. Such fluorescent probes are known for a widevariety of species, including sodium, potassium, calcium, magnesium,zinc, chloride, phosphate and oxygen [41-50]. Hence, visual sensors canbe anticipated for a wide range of analytes.

EXAMPLE 4

Visual Polarization Sensing Using the Same Substance as the Reference

In this example, we show how the measurement can be accomplished usingthe same fluorophore as the sample and as the reference. Thispossibility was discussed above (FIG. 13 and FIG. 1) where we describedthe use of an additional polarizer in front of the sample to select theperpendicular component of the sample emission. The optical arrangementis shown in FIG. 11 (top). In this case the light source was anelectroluminescent device which was powered by a 9 volt battery. Theoutput from the ELL was blue and centered near 480 nm.

To illustrate this method of visual sensing we used a sample whichcontained varying concentrations of [Ru(bpy)₃]²⁺. Such metal ligandcomplexes are now being widely used as luminescent probes whencovalently attached to macromolecules [51-55] and as chemical sensors[56-61]. As the reference we used the same fluorophore [Ru(bpy)_(3]) ²⁺at a constant concentration. The emission from this reference sample wasviewed through a polarizer (P_(⊥)) to select just the perpendicularcomponent at its emission. The combined emission from the sample andreference was then viewed through the dual-polarizer analyzer-polarizercombination. The optical arrangement is shown on the top of FIG. 11.

The compensation angles for various concentrations of [Ru(bpy)₃]²⁺ areshown in FIG. 11. These angles are the differences between the polarizerangles needed to equalize up the intensities in the absence and presenceof the indicated [Ru(bpy)₃]²⁺ concentration. These results demonstratedthat visual polarization sensing is possible using the same fluorophoreas the reference. This approach has the advantages of automaticallyproviding the same visual wavelengths for the sample and reference, andthereby avoiding any difference in color on each side of the dualpolarizer.

It should be noted that these results suggest the use of visualpolarization sensing in a number of common applications. Luminescentmetal ligand complexes have been used in immunoassays [53-54], so thatone can anticipate visual immunoassays based on intensity changes ofmetal-ligand complexes. Visual immunoassays may also be possible withthe usual fluorophores with nanosecond decay times. Changes in theluminescence intensity can be caused by energy transfer or a variety ofother mechanisms [62-63]. Another possible application is for industrialor household determination of oxygen, pH or salt concentrations.Metal-ligand probes are known which are sensitive to oxygen [56-57] orpH [60], and probes are known which are quenched by chloride [48-49].Hence, one could develop visual sensors for these common analytes.

EXAMPLE 5

In this example, the sensing of glucose and calcium using the FIG. 16apparatus is described. In FIG. 16, the reference and sensing moleculesare housed in containers 10 and 11, respectively. Containers 10 and 11have associated therewith a vertical and horizontal polarizer film,respectively. The excitation light travels from the source (not shown)through filter 12, then through containers 10 and 11, then throughfilter 13, then finally through analyzer polarizer 14. The analyzerpolarizer 14 may be rotated until the adjacent intensities from thevertically and horizontally polarized emission are equalized. The angleof the analyzer polarizer is used to determine the analyteconcentration.

The glucose assay was accomplished using the glucose/galactose bindingprotein from E. Coli (GGBP). We used a mutant which contained a singlecysteine residue at position 26 (Q26C GGBP) [19]. This protein waslabeled with (4′iodoacetamidoanilino)-naphthalene-6-sulfonic acid(I-ANS) from Molecular Probes, Inc. A solution containing 2.5 mg/ml Q26CGGBP in 20 mM phosphate, 1 mM tris-(2-carboxyethy)phosphine (TCEP), pH7.0 was reacted with 50 μL of a 20 mM solution of I-ANS intetrahydrofuran (purchased from Molecular Probes, Inc.). The resultinglabeled protein was separated from the free dye by passing the solutionthrough a Sephadex G-25 column. The protein-ANS conjugate was purifiedfurther on Sephadex G-100 and dissolved in 20 mM phosphate, pH 7.0. Thelabeled protein was used as both the reference and the sensing molecule.

The calcium assay was performed using Fluo-3 was obtained from MolecularProbes, Inc. as both the reference and the sensing molecule. The calciumconcentration was controlled using the calcium buffer kits, C-3009, alsofrom Molecular Probes, Inc.

The images seen through the analyzer polarizer are shown in FIG. 17. Forthese images the initial angular rotation of the analyzer (α₀) wasadjusted to match the intensities seen from both sides of the sensor.The images were then recorded for various analyze concentrations, withthe analyzer left in the same angular position (α₀) . As the glucoseconcentration increases the intensity from the horizontal (right) sideof the sensor decreases, as can be seen in the top panel of FIG. 17. Asthe calcium concentration increases the intensity in the horizontal(right) side of the sensor decreases, as can be seen in the lower panel.

The angular position of the analyzer can be adjusted to yield visuallyequivalent intensities. These angles are shown under the images in FIG.17. For glucose the angles of the analyzer must be increased to equalizethe intensities. This effect is due to the decreased horizontalintensity, and the need to increase the horizontal intensity to visuallymatch both sides of the sensor. For increasing concentrations of calciumthe angle of the analyzer must be decreased to equalize the intensities.This direction of change is needed because the increased intensity fromthe horizontal component must be attenuated to yield visually equivalentintensities.

FIGS. 18 and 20 show the calibration curves for the compensation anglesfor glucose and calcium, respectively. We have found that thecompensation angles are typically accurate to 1 or 2 degrees [64]. Anaccuracy of 1 degree in the compensation angle results in an accuracy of±0.5 μM in glucose and ±0.05 μM in calcium, as found for the mostsensitive part of the curve. While the accuracy is somewhat less thanavailable with electronic detection, the accuracy may be adequate forsome clinical or analytical applications, particularly where a yes/noanswer is adequate.

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We claim:
 1. A method for determining the presence or concentration ofan analyte in a sample, comprising the steps of: a) providing afluorescent reference molecule and a fluorescent sensing molecule; b)exposing said sensing molecule to a medium containing an analyte to forma mixture, wherein said analyte is capable of changing the intensity ofthe fluorescence emitted by the sensing molecule in aconcentration-dependent manner; c) exposing said reference molecule andsaid mixture to a radiation source which causes said reference andsensing molecules to emit fluorescence; d) polarizing said emittedfluorescence along two different polarization axes which aresubstantially perpendicular to each other; e) attenuating the emissionfrom one of the polarization axes, if necessary, such that theintensities of the emissions through both axes are substantially equal;and f) correlating the degree of attenuation with the presence orconcentration of said analyte in said sample.
 2. The method of claim 1,wherein the determination of emission intensity in step e) is performedvisually.
 3. The method of claim 1, wherein the determination ofemission intensity in step e) is performed electronically.
 4. The methodof claim 3, wherein the electronic determination of emission intensityis performed by independently measuring the two intensities.
 5. Themethod of claim 3, wherein the electronic determination of emissionintensity includes measurement in a balancing circuit.
 6. The method ofclaim 3, wherein the electronic determination of emission intensityincludes measurement of the ratio of the two intensities.
 7. The methodof claim 1, wherein step d) is performed by allowing the emittedfluorescence to pass through a dual polarizer.
 8. The method of claim 1,wherein the polarized emission is passed through a rotatable polarizer;wherein the attenuation is performed by rotating said polarizer; andwherein said correlation includes determining the amount of rotation ofsaid polarizer.
 9. The method of claim 1, wherein the fluorescentreference molecule is attached to a film.
 10. The method of claim 9,wherein the film is a stretched polymeric film.
 11. The method of claim1, which further comprises filtering said emitted fluorescence tosubstantially eliminate scattered excitation.
 12. The method of claim 1,wherein the reference molecule and the sensing molecule are distinctentities having the same structure, and the reference molecule isisolated from the analyte.
 13. The method of claim 1, wherein theanalyte is selected from the group consisting of oxygen, glucose, bloodgases, proteins and ions.
 14. A sensor for determining the presence orconcentration of an analyte in a sample, which comprises: a) means formaintaining a fluorescent reference molecule; b) means for maintaining afluorescent sensing molecule, wherein said analyte is capable ofchanging the intensity of the fluorescence emitted by the sensingmolecule in a concentration-dependent manner; c) optionally a radiationsource which is capable of causing said reference and sensing moleculesto emit fluorescence; d) means for polarizing said emitted fluorescencealong two different polarization axes which are substantiallyperpendicular to each other; and e) means for attenuating the emissionfrom one of the polarization axes, such that the intensities of theemissions through both axes are substantially equal.
 15. The sensor ofclaim 14, which further comprises electronic means for the determinationof the intensity of said emitted fluorescence.
 16. The sensor of claim14, wherein component d) comprises a dual polarizer.
 17. The sensor ofclaim 14, wherein component e) comprises a rotatable polarizer.
 18. Thesensor of claim 14, wherein the fluorescent reference molecule isattached to a film.
 19. The sensor of claim 18, wherein the film is astretched polymeric film.
 20. The sensor of claim 14, which furthercomprises means for filtering said emitted fluorescence to substantiallyeliminate scattered excitation.
 21. The sensor of claim 14, wherein theradiation source is external to the sensor.
 22. The sensor of claim 21,wherein the light source is selected from the group consisting ofsunlight and room light.